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Three different methods to use Platelet-rich plasma in the treatment of skin
wounds in equine distal limb
Roberta Carneiro da Fontoura Pereira, Flávio Desessards De La Côrte
Title Page 1
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Three different methods to use Platelet-rich plasma in the treatment of wound in equine 3
distal limb 4
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Roberta Carneiro da Fontoura Pereira *, Flávio D De La Côrte*, Karin E Brass, Marcos da
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Silva Azevedo* Camila Cantarelli*, Miguel Gallio*, Alfredo Skrebsky Cezar§, Maria Andréia
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Inkelmannǂ
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* Department of Large Animal Clinics, School of Veterinary Medicine, Federal University of 10
Santa Maria, Santa Maria, Rio Grande do Sul, Brazil
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§ Department ofPreventive Medicine, School of Veterinary Medicine, Federal University of
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Santa Maria, Santa Maria, Rio Grande do Sul, Brazil
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ǂ Department of Agrarian Studies in Regional State University of Northwestern Rio Grande do
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Sul (UNIJUÍ), Brazil
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Correspondence: Roberta Carneiro da Fontoura Pereira, Department of Large Animal Clinics,
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School of Veterinary Medicine, Federal University of Santa Maria, Santa Maria, Rio Grande
20 do Sul, Brazil, 97.105-900 21 E-mail: betacfp@hotmail.com.br 22 Tel.: +55 55 9935 3511. 23
SUMMARY
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Reasons for performing the study: Distal limb wounds are frequent in horses after traumatic
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events. The use of platelet-rich plasma (PRP) represents a simple method of treating wounds in
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equines because the platelets contain several growth factors.
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Objectives: To evaluate the clinical effect of autologous PRP on wound healingand to establish
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a correlation between the number of platelets in PRP and healing time of wounds and individual
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variations in healing time of wounds,regardless of treatment group. In addition, to determine
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what the best form of application of PRP equine skin wounds and the test of using the
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homologous PRP whit alternative as a treatment alternative in cutaneous wounds of horses.
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Study design: This study evaluates the potential of platelet derived factors to enhance wound
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healing in the lower equine limb.
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Methods: To test this hypothesis, eight healthy adult horses, 8± 3.76 years old were used. Four
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skin wounds of 4cm2 area were surgically created in the dorsolateral aspect of the third cannon
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bone, two on the left forelimb (A1 and A2) and two on the right forelimb (A3 and A4). The
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cutaneous lesions were treated according to the randomly established treatment group. In the
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same animal, each of the four wounds was assigned to one of four treatment groups at random,
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for each treatment group obtained eight replicates in different regions in the forelimbs. In the
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group (G) I, the skin lesions were treated with PRP infiltration of the wound edges; GII were
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treated with PRP was used in gel form; GIII was used homologous PRP infiltration and GIV
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control group (without PRP application).
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Results: PRP gel treatment (GII) reduced in 15 days the healing time compared to the wounds
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of the control group (GIV). The GII presented higher frequency of low inflammation and
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neovascularization low to moderate in biopsies 1 and 2 on the histopathology exam. The GIV
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had the highest intensity of granulating wounds between the groups.
Conclusions: PRP in gel form, showed the best result on wound healing in the distal limb of
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horses in both clinical and histopathology evaluations. Furthermore, homologous PRP can be
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considered beneficial in the management of wound healing in horses and it can be safely used
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as an alternative source of PRP.
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INTRODUCTION
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Wounds resulting from environmental disorders are common causes of substantial
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economic losses in equine veterinary medicine [1], because the wounds require intensive care
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and expensive treatments [2]. Factors with size of the wound, tension, mobility, poor blood
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supply, susceptibility to infection, and type and condition of underlying tissues of the wound
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are directly interfere with healing [3]. The cellular mechanisms as described in repair distal
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limb soft tissue, such as, inefficient inflammatory response to trauma, persistent local up-
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regulation of pro-fibrotic growth factors, a disparity between collagen synthesis and lysis as
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well as microvascular occlusion and deficient apoptosis of the cellular components of
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granulation tissue and a site very favorable to contamination [4, 5 ].
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Horses often suffer from chronic slow healing wounds and, conversely, from the
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development of exuberant granulation tissue, when soft-tissue wounds are located on the distal
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limb. Therefore, wounds occurring in this location are of important concern to horse owners
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and veterinarians [6].
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Several clinical studies, in both human and veterinary medicine, on the restoration of
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tissue integrity have shown the positive role of platelets in natural wound healing [7, 8, 4, 9,
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10]. When locally applied, platelets accelerate healing of normal tissue because of growth
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factors release, as already widely demonstrated [11]. Platelet-rich plasma (PRP) has been
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utilized as a source of cytokines and growth factors (GF) released from activated platelet alpha-
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granules at the site of tissue injury. GF’s released after platelet degranulation include
transforming growth factor β (TGF- β), platelet-derived growth factor (PDGF), insulin-like
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growth factor-I, vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)
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[12].
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The aim of this study was to evaluate the clinical effect of autologous PRP on wound
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healingand to establish a correlation between the number of platelets in PRP and healing time
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of wounds and individual variations in healing time of wounds,regardless of treatment group.
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In addition, to determine the best form of application of PRP on equine skin wounds and the
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possibility of using homologous PRP as a alternative treatment in cutaneous wounds of horses.
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MATERIAL AND METHODS
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Selection of Animals: This study was approved by the Committee of Ethics on Animal Care
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and Research Use of the Federal University of Santa Maria (UFSM) registration number
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110/2013. This study used eight healthy adult horses, geldings and females, with an average
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age of eight years (± 2.6), average weight of 500 kg (±70), with normal CBC and biochemical
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parameters within the reference values, including platelet counts above 100.000/dL. The
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animals were kept in individual stalls fed daily with commercial diet, alfalfa hay and water ad
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libitum. The animals received preoperatively flunixin meglumine (1.1 mg/kg intravenous (IV),
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once a day) for 3 days, penicillin-based antibiotics 22.000 IU/kg (procaine penicillin,
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benzathine, and dihydrostreptomycin) single intramuscular application (IM) and tetanus
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antitoxin 5.000 UI via IM. After induction of cutaneous defects, the site was protected with
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bandages, changed daily until end of the study (complete healing of wound).
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Preparation of Platelet-Rich Plasma (PRP): Preparation of Platelet-Rich Plasma was
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performed using 450 ml of blood were collected from each animal by puncturing the external
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jugular vein using a commercial blood-transfusion bag containing citrate-phosphate-dextrose
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solution with adenine as an anticoagulant (CPDA-1; 63 mL for 450 ml of blood). Then, the PRP
is obtained from 100 ml of whole blood aspirated from the blood bag and distributed in three
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polypropylene tubes falcon type (50ml) where in each tube were deposited 30ml and other two
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35 ml. This tubes were centrifuged a first time at 224 g for 10 minutes to separate the plasma
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from the mist zone (phlogistic layer containing leukocytes) and red blood cells. The supernatant
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containing the platelet-rich plasma was transferred to a second falcon tube and centrifuged at
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440g will again for 10 minutes. After the second centrifugation, the plasma supernatant was
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discarded, and preserved only 10 ml of PRP, representing 10% of the initial total blood volume.
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This technique to obtain PRP was previously determined by Pereira et al. [13].
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Preparation of homologous PRP: PRP homologous was obtained through the blood collection
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from the two donor horses and processed according to the protocol proposed by Pereira et al.
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[13]. The wounds of animal 1, 2, 3 and 4 was used homologous PRP of blood donor 1.The
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wounds of animal 5, 6, 7 and 8 has used homologous PRP of blood donor 2. Laboratory tests
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were realized in the previous period to the beginning of the experiment to rule out equine
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infectious anemia and glanders.
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PRP activation for preparation the PRP in gel form: For activation of platelets, calcium
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gluconate was used to make PRP in the gel form. For each ml of PRP to be transformed into
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gel, 0.5 ml of the supernatant plasma it was removed and it is considered as the platelet poor
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plasma (PPP). Then, 0.3 ml of calcium gluconate was added to this PPP and incubated at 37 °
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C for 15 minutes. After the incubation period, resuspension was performed thrombin and
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subsequent centrifugation at 640 g for 10 minutes at room temperature [14]. The mixture of
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autologous thrombin with PRP condense further with formation of PRP in gel form that was
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applied in the injuries destined to group treated to PRP gel.
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Creating the skin defects: All horses received a dose of 10% xylazine (1mgkg-1) and
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buthorfanol tartarate (0.02mg/ kg prior to the surgical procedure. The high palmar nerve block
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was performed with the technique described by Stashak & Baxter [15]. With the horses
standing, two cutaneous defects were created on the dorsolateral aspect of each McIII, the first
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defect located 3cm below the carpometacarpal joint and the second defect 3cm above the fetlock
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level. Distance between the two wounds in each forelimb created was 5 cm. To create the skin
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defects, the skin was clipped and prepared for aseptic preparation with iodine povidine and 70%
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alcohol. Sterile gloves were used to decrease the likelihood of contamination. A plastic frame
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of 4cm2 was place over the site to be incised and with the aid of a 24 scalpel blade, the skin was
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removed leaving a 4 cm2 defect on the skin, one defect proximal and the second defect distal,
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over the dorsolateral region of McIII. Once the cutaneous defects were created, one treatment
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was randomly assigned to each wound. A sterile bandage was placed over the wounds, for
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protection, which was changed daily.
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Treatment Groups: Each skin defect was considered a treatment group and each group
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received three applications of PRP, repeated applications every 2 weeks, as follows:
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1. GI: PRP infiltration subcutaneously around the wound edges.
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2. GII: Skin defects were treated with PRP gel form over the skin defect.
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3. GIII: PRP Infiltration homologous subcutaneously around the wound edges.
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4. GIV: Infiltration of saline around the wound edges was used as a control group.
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A total of 32 skin wounds were randomly treated and distributed in the four wounds of
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the eight animals evaluated (Figure 1).
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Clinical evaluation: The clinical evaluation was blinded and performed by three veterinarians,
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every 15 days, using the following parameters: presence of granulation (low, moderate,
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excessive), color (pale, pink and hyperemic) and sensibility (absent or present). The size of the
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wound was measured using a tape measure.
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Histopathological analysis: The skin biopsies were performed on the 15th and 30th day after
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the first application of PRP. The animals were sedated with 10% xylazine at a dose of 1 mg /
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kg and received local anesthesia with 2% lidocaine without vasoconstrictor. Biopsy samples
were collected with a 8mm-dermatological punch under aseptic preparation. In the first sample
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each treatment group was collected from the upper right and lower left corner of the wounds
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and the second biopsy was taken in the upper left and lower right corner of the wounds. Samples
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were immediately fixed in 10% formalin. In laboratory the samples were then routinely
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processed for histopathology, prepared and stained by hematoxylin and eosin (HE). The
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variables analyzed were: intensity of inflammation, granulation tissue, fibroplasia, fibroblasts
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cells, neovascularization and collagen maturity, on a scale of low, moderate and intense (Table
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1).
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Statistical analysis: The number of platelets in whole blood and number of platelets in PRP
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were submitted to the distribution normality test by Kolmogorov-Smirnov test, with 95%
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confidence. After verified the normal distribution (p = 0.1208), Pearson’s correlation was tested
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between these variables at a 95% confidence interval. The average of three evaluations of the
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variables number of platelets in whole blood and number of platelet in PRP and the variable
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healing time wounds control and treated PRP infiltrated, PRP gel and homologous PRP were
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submitted to the Kolmogorov-Smirnov normality test with 95% confidence intervals (p> 0.05).
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Pearson's correlation was analyzed between the average of the number of platelets in whole
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blood of animals and the healing time of each type of wound, as well as between the average
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number the platelets in PRP animals and time healing for each type of wound. The data were
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statistically analyzed using the chi-square test and Fisher's exact test, with a 95% confidence
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interval, considering as categorical variables and whether or not treatment with PRP and
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evaluating the healing time of frequencies lower, equal or higher than expected. The healing
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time was evaluated by comparing (ANOVA) and comparing the groups by Tukey test with 95
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% confidence interval (p ≤ 0.05). The proportion of animals with healing time than the median
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of the control group was evaluated for each group treated by Fisher's exact test with 95 %
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confidence interval (p ≤ 0.05). The variables intensity of inflammation, granulation tissue,
fibroplasia, fibroblast cells, neovascularization and collagen maturity were compared using
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Fisher's exact test with 90% confidence interval (significance level of p ≤ 0.1).
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RESULTS
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Platelet-Rich Plasma 174
Positive correlation was found between the number of platelets in whole blood and
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platelet number in PRP, with (p <0.0001) (Figure 1).
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There was no correlation between the number of platelets in the whole blood of horses
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and the healing time. In addition, no correlation between the average number of platelets in
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PRP of animals used and the healing time of groups I, II, III and IV.
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Clinical evaluation 180
Considering treatment with PRP gel, the healing time was reduced fifteen days when
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compared to the control group in 62.5% of horses (animals 1, 2, 4, 5 and 6) (p = 0.007). Only
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the animal 8 there was an increase of healing time. Thus, it is clear that PRP treatment allowed
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a faster healing in most cases. The homologous PRP treatment reduced the time to healing of
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the injured animal 3, 7 and 8 (37.5%) compared to control group, and in another animals the
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healing time of the wound was the same as control group. Wounds treated with PRP infiltration
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was reduced fifteen days the healing time compared to control wounds only in the animal 2 and
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5 (25.0%) and increased in fifteen days the healing time in the animal 8. Regardless of treatment
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used, different found in healing time between the evaluated animals.
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Histopathological evaluation 190
In the analysis of variables intensity of inflammation, granulation, fibroplasia, fibroblast
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cells, neovascularization and collagen maturity it was observed that the group GIV (control)
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had higher frequency of the intensity the formation of granulation the GII (PRP gel), followed
GIII (homologous PRP) and GI (PRP infiltration), however, the GII showed a higher frequency
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of low inflammation on biopsy 2 compared to GIV. When comparing all groups and between
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first and second biopsies, GII showed most low to moderate frequency of neovascularization.
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In did No observe differences were observed regarding fibroplasia, fibroblast cells and collagen
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maturity and between the first and second biopsies (Table 1).
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DISCUSSION
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Wounds located on the distal limb in horses have a very important characteristic, a
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chronic nonhealing wounds and exuberant granulation tissue often develop [16].
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Distal limb lacerations, with or without tissue loss, represent a challenging situation to
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veterinarians to repair them by first intention, leaving the tendons/tendon sheaths and joints
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unprotected. Associated to this lack of tissue coverage, the wound located on the distal limb.
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This study served to fulfill a gap in the literature, due to the fact that evaluated three
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different methods of PRP treatment to shorten the healing time and to obtain a better healing.
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The surgically created wounds on the dorsolateral aspect of the cannon bone and treated with
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three applications of the PRP demonstrated that PRP in gel form was able to reduce in fifteen
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days the healing time when compared to control group.
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It was observed that difference healing in horses evaluated, regardless of treatment
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group, no correlation was found between the number of platelets in the PRP and the healing
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time of wounds.
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Maciel et al. [9] compared one or two applications of platelet-rich plasma gel in burn
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wounds of horses and observed an increased speed of repair of the extracellular matrix and its
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components in deep second degree burn, but had a potential of fibroses formation. They
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concluded that two topical applications of the PRP gel accelerated the extracellular matrix
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formation during the first half of wound healing. One experimental study conducted by
Monteiro et al. [17], with topical application of autologous platelet-rich plasma gel, did not see
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acceleration or improvement in the quality of repair of small granulating wounds on limbs of
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horses when compared to the control group.
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Reviewing published data, one will discover that there is a lack of information about the
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use of the homologous PRP for the treatment of cutaneous lesions in horses. Barbosa et al. [18]
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consider hypovolemia and thrombocytopenia as limiting factors for the obtaining of PRP. One
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of the hypotheses of this study was to evaluate the possibility of homologous whole blood for
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obtaining PRP, alternative source for growth factors when faced with extensive skin lacerations
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where animals, due to injury, lose considerable amounts of blood. Another situation would be
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when dealing with neonatal foals, where we can't remove large volumes of whole blood for
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obtaining autologous PRP. 228
Carmona et al. [19] described a clinical case of a chronic severely contaminated distal
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limb wound in an 8-month-old foal, treated with debridement, bandages and PRP (gel form and
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infiltration), this authors encouraged to use of PRP, especially in large wounds. According to
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the results presented in their study, the homologous PRP is a viable form of use of PRP in
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equine skin wounds.
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In the present study, there were no differences in fibroplasia variables, fibroblast cells
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and collagen maturity, between the groups GI, GII and GIII). The treated group with PRP gel
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(GII) showed a low to moderate frequency the neovascularization and inflammation in the first
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and second biopsies, and low frequency of the granulation tissue when compare with group
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control. Carter et al. [4] evaluated platelet derived factors to enhance wound healing in the
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lower equine limb; the cutaneous lesions treated with PRP gel accelerated epithelial
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differentiation and tissue organized collagen bundles. Vendramin et al. [20] compared the use
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of PRP infiltration and PRP gel form for treatment of experimental skin wounds in rabbits;
these authors observed an increase in the inflammatory response and the intensity of collagen
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production, an increase in the number macrophages and fibroblasts in the graft site.
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CONCLUSIONS
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The number of platelets in the PRP did not influence the cutaneous healing horses.
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Based on clinical and histopathological evaluation, the PRP gel was the best form to use it for
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treatment of skin wounds in horses. The homologous PRP can be used as an option for the
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treatment of wounds healing in horses.
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TABLE LIST
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Table 1 - Descriptive results of biopsy in the different groups.
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Biopsy Intensity Group I Group II Group III Group IV Inflammation intensity First Low 1 1 1 0 Moderate 3 3 4 2 Intense 4 4 3 6 Second Low 1 4 1 0 Moderate 3 3 3 6 Intense 4 1 4 2 Granulation tissue First Low 0 0 0 0 Moderate 5 3 5 4 Intense 3 5 3 4 Second Low 0 2 0 0 Moderate 4 4 4 4 Intense 4 2 4 4 Fibroplasia First Low 0 1 0 0 Moderate 4 3 5 3 Intense 4 4 3 5 Second Low 0 2 0 0 Moderate 4 4 4 4 Intense 4 2 4 4 Fibroblast cells First Low 0 1 0 0 Moderate 4 3 5 4 Intense 4 4 3 4 Second Low 0 2 0 0 Moderate 4 4 4 4 Intense 4 2 4 4
Neovascularization First Low 0 2 1 1 Moderate 4 6 5 3 Intense 4 0 2 4 Second Low 1 4 0 0 Moderate 4 0 4 3 Intense 3 4 4 5 Collagen maturity First Low 6 2 3 5 Moderate 2 4 5 2 Intense 0 2 0 1 Second Low 5 3 4 6 Moderate 2 3 4 2 Intense 1 2 0 0 266 267
LIST OF FIGURE LEGENDS
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Figure 1 -Schematic design of the random distribution of treatments in skin wounds. MAE:
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left forelimb (A1 and A2); MAD: right forelimb (A3 and A4) the treatment of wounds in
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animals.
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Figure 2 - Simple linear regression and Pearson’s correlation between number of platelets in
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whole blood and number of platelets in PRP obtained from horses.